Molecular basis for gating of cardiac ryanodine receptor explains the mechanisms for gain- and loss-of function mutations

Cardiac ryanodine receptor (RyR2) is a large Ca2+ release channel in the sarcoplasmic reticulum and indispensable for excitation-contraction coupling in the heart. RyR2 is activated by Ca2+ and RyR2 mutations are implicated in severe arrhythmogenic diseases. Yet, the structural basis underlying channel opening and how mutations affect the channel remains unknown. Here, we address the gating mechanism of RyR2 by combining high-resolution structures determined by cryo-electron microscopy with quantitative functional analysis of channels carrying various mutations in specific residues. We demonstrated two fundamental mechanisms for channel gating: interactions close to the channel pore stabilize the channel to prevent hyperactivity and a series of interactions in the surrounding regions is necessary for channel opening upon Ca2+ binding. Mutations at the residues involved in the former and the latter mechanisms cause gain-of-function and loss-of-function, respectively. Our results reveal gating mechanisms of the RyR2 channel and alterations by pathogenic mutations at the atomic level.

Line 304: "However, since S6cyto still restricts the U-motif, the activity may be medium (Fig. 6b, right)." I assume that "medium" is equivalent to the level of opening of WT? This should be pointed out.
Line 306: "Upon Ca2+ binding, conformational changes stop on the way, resulting in loss-of-functon (Fig. 6c,right)." Stopping on the way sounds odd, please improve.
Lines 318-328. In this paragraph of the discussion, the open structure in the presence of only Ca2+ is compared to the open structures with Ca2+ and either PCB95 or caffeine added. The paragraph is purely descriptive and would be enhanced with discussion on at least what could be the effect of caffeine, whose location is known. The sentence "Ca2+ 326 /PCB95 open structure was only determined in the absence of FKBP12.6; addition of FKBP12.6 closes the channel" is confusing; please re-write.
Lines 346-354: The stopper feature should be re-defined here in the discussion. The manuscript by Kobayashi et al. studied the gating mechanism of RyR2 by Ca2+. The authors presented two high-resolution structures of mouse RyR2 in both closed and open states and used many point mutations to test their hypothesis based on whether the mutations caused gain-or loss-offunction. The authors described two independent pathways for RyR2 gating. The first involves interactions close to the channel pore, which are destabilized upon Ca2+ binding. The second pathway include a series of conformational transductions from U-motif to S4-S5 linker, which moves out of the way and opens the gate. This is the first paper to solve the full-length mouse RyR2 structure in both closed and Ca2+ bound open states, both WT and K4593A mutant. This is also the first paper trying to explain the gating of RyR2 in detail using structural approach. The study is important and meaningful; the manuscript is well-written, but I feel that some clarification is needed and some small mistakes need to be corrected. Specifically: 1. Could the authors explain in more detail about what is the cause of the U-motif compaction? In my understanding, the direct conformational changes upon Ca2+ binding are in the CTD since it contains the Ca2+ binding site. The only interaction between CTD and U-motif seems to be the hydrophobic interaction between F4888 in the CTD and the hydrophobic pocket in the U-motif. However, in K4593A mutant, there is no U-motif compaction/rotation. The authors described the CTD/U-motif/S6cyto complex to be "tightly attached to each other" (line 108). If so, why in the K4593A mutant there is no rotation of the complex but rather only the CTD is moved after Ca2+ binding? The authors explained that the K4593-S4167 interaction is important for U-motif compaction. It seems to me that the interaction is not the cause of the compaction, instead, it stabilizes the compaction. Could the authors describe how the conformational changes transduce from Ca2+ binding domain of the CTD to the Umotif compaction? 2. In data method, the authors described the data collection and image processing of the mutant F4888A. However, the authors showed no structure of F4888A in the paper. And the data collection and image processing details for K4593A is missing in the Methods section. This needs to be corrected.
3. With regarding to F4888A, I think it is actually very interesting to see the structure if the authors indeed collected and processed the data. To my understanding, this mutation will abolish the interaction between CTD and the U-motif, which means it will stop the conformational transduction from CTD downstream. The result, I imagine, should be similar to the structure of K4593A where only the CTD moved upon Ca2+ binding. However, functional study proved that it is actually a GOF mutation. Could the authors explain in more detail how the destabilization of CTD-Umotif interaction causes GOF? Are there other interactions between CTD and the U-motif? If the authors can show more interaction between CTD and U-motif that would be really helpful to understand the GOF of F4888A mutant and the alanine-substitutions of the neighboring residues that forms the hydrophobic gate in the U-motif. 4. In Fig6, for panel A legend, I believe the authors made a mistake stating that "Yellow and dark blue arrows indicate interactions only found in the closed and open states, respectively". Purple arrows seem to indicate closed-state-only interactions while green arrows seem to indicate open-state-only interactions. Also, could the authors show and discuss the case of the alanine-substitution of F4888 and neighboring residues in panel D? 6. In movie S4, D4715 is mislabeled as "D4751". 7. Whenever we encounter a LOF mutation, there is always a possibility that the channels are misfolded/did not go to the membrane. Could the authors prove that all the LOF mutants that did not show any signal in the ryanodine binding assays are correctly folded channels? If not, could the authors explain why they think it is not necessary or not possible prove it? 8. Could the authors explain why different 3D classes were found during Cryo-EM data processing if "no major differences were observed among the classes"? (Line91) 9. It would be helpful if the authors could show the distances between atoms when highlighting interactions in atomic models.
10. The authors hypothesized that there are two independent pathways for channel opening. If the authors could make a double GOF mutant from each of the pathways (e.g. D4744A and V4879) and use functional experiments to show that there is an additive effect or even spontaneous channel opening, that would be interesting and would make the argument stronger.
in the overall movement were added for the understanding to readers less familiar in the field.

2.
Density maps for the most relevant residues in different 3D reconstructions coming from the same conditions were attritionary presented in the Supplementary Figures 3b, 4d, h, 6a.
4. Detailed description about K4593R causing LOF, in spite that K to R is thought to be generally mild mutation, was added. Figure 3o to explain the role of a "stopper" formed by the interaction of L4505-F4749, and detailed description about the "stopper" formed by the interaction of L4505-F4749 was added.

Modification of
6. Detail description for an explanation of the mechanism, by which U-motif compaction occurs, was added.  MAIN CONCERN This is a highly specialized subject of a protein that does not benefit from the extensive "common knowledge" that other ion channels have. In addition, it is a huge channel with many domains, and the jargon is unavoidable. Therefore, for each results section I would recommend adding few sentences to offer a parallel, easier lecture to readers less familiar in the field.
Thank you for your kind advice. At the beginning of each results section, we added some explanations to clarify which part we describe in the overall movement (lines 158-159, 181-182, 214, 262-263).

SPECIFIC POINTS
The validation files show a high clash score. Although this does not affect the region being examined, with the resolution level it should be possible to achieve better statistics.
Thank you very much for your point out. We deeply understand the importance of the achievement of better statistics. We notice that clashscore of our data is increased by a low resolution of a large N-terminal cytoplasmic region, which is not focused on in this paper (Supplemental Fig. 1f,g,8a,b). Therefore, we calculated the clashscore for the core domain by excluding the cytoplasmic region and found that they became lower than those for the whole molecule. https://pubmed.ncbi.nlm.nih.gov/33404525/), clashscore around 10 is not so high among 2,750 molecules deposited in the PDB that have map resolutions of better than 5 Å (see Fig. 6a in the paper). We will continue to make efforts to achieve better statistics and will update the PDB as soon as they are corrected. We appreciate your constructive suggestion. We now added supplementary figures illustrating the reproducibility of their density maps for the most relevant residues in different 3D reconstructions coming from the same conditions ( Supplementary Fig. 3b, 4d, 4h, 6a).
The 3D mutant that has been determined is K4593A, however the mutant described in methods is F4888A We apologize for this mistake. It is now corrected (line 499).
The "S2-S3 domain" term is confusing as it includes two TM helices. It would be more accurate to refer to it as S2-S3 linker domain or a similar term.
We changed "S2-S3 domain" to "S2-S3 linker domain". Line 133 "Two pathogenic mutations, K4593Q and K4593R, also led to loss of binding." The K to R mutation is very conservative which illustrates the subtlety of RyR's domain interfaces.
It would be helpful that the authors elaborate/discuss this unexpected LOF effect.
Thank you very much for your indication. As you pointed out, K to R mutation is generally thought to be conservative mutation. However, in K4593R, two nitrogen atoms (N h1 and N h2) at the tip of Arg might occupy both oxygen atoms (O e1 and O e2) from E4198 in the open state, which prevents S4167-E4198 interaction essential for the U-motif compaction and the channel opening.
Therefore, it is reasonable that K4593R exhibited similar behavior as observed in K4593A.
To explain these, we added a sentence "In K4593R, two nitrogen atoms (N h1 and N h2) Fig. 3b)." In this sentence, the term "interacting partners" may evoke interaction with other protein partners; please improve sentence.
We revised the term "interacting partners" to "the interacting pairs" (line 139).
Lines 152, section "Movements of the S1-S4 bundle lead to outward movement of the S4-S5 linker" The finding that mutations affecting the interactions between different helices of the voltage-sensor like domain can either activate or inhibit is unexpected but real. The authors should expand on the differential role of the helical bundle helices; currently it is not very clear.
We changed the description to clearly explain the differential role of the TM helices by adding a new figure (Fig. 3o) Fig. 4o, Supplementary Fig. 4g), we assume that the rewinding to α-helix in the upper part of S4 provides a margin for shortening of the S4-S5 linker. Therefore, it is reasonable that the stopper (L4505-F4749 interaction) is required to stabilize the unwinding form of the upper part of S4 and S4-S5 linker.
To explain these, we replaced Fig. 3o with the new one. In addition, the following sentences are added to the main text. "Since α-helix formation of the S4-S5 linker shortened its length in the open state (Fig. 3o, Supplementary Fig. 4g), we assume that the rewinding to α-helix in the upper part of S4 provides a margin for shortening of the S4-S5 linker." (lines 205-208)

Line 300: "In addition, S6cyto restricts movement of the U-motif toward the S2-S3 domain. "
It is counterintuitive that S6cyto affects the U-motif, as it appears that S6cyto is both the cause and the consequence of the movement of the U-motif. Please explain better.
We changed it to "In addition to direct regulation of the channel gate, S6cyto interacts with Umotif to restrict its movement toward the S2-S3 linker domain (Fig. 6b, left)." (lines 327-329).
Line 304: "However, since S6cyto still restricts the U-motif, the activity may be medium (Fig. 6b,   right)." I assume that "medium" is equivalent to the level of opening of WT? This should be pointed out.
We changed it to "Since the interaction still maintains at the open state, the WT channel is not fully activated". (lines 330-331).
Line 306: "Upon Ca2+ binding, conformational changes stop on the way, resulting in loss-offunction (Fig. 6c, right)." Stopping on the way sounds odd, please improve.
We changed it to "This interrupts Ca 2+ -induced conformational changes, resulting in loss-of- We changed it to "In the Ca 2+ /ATP/caffeine structure, in contrast, both CTD, U-motif and S2-S3 domain made a translational outward movement toward caffeine ( Supplementary Fig. 10a,
We changed it to "Ca 2+ /PCB95 open structure was only determined in the absence of FKBP12.6, because the addition of FKBP12.6 closes the channel." (lines 353-354) Lines 346-354: The stopper feature should be re-defined here in the discussion.
We described that "We showed that the corresponding salt bridges between R4501, Y4720, and D4744, which are critical to keep S4 in place for appropriate positioning of the stopper (L4505- We apologize for this mistake. It is now corrected to purple and green.

Signed by Montserrat Samso
Reviewer #2 (Remarks to the Author): The manuscript by Kobayashi et al. studied the gating mechanism of RyR2 by Ca2+. The authors presented two high-resolution structures of mouse RyR2 in both closed and open states and used many point mutations to test their hypothesis based on whether the mutations caused gain-or loss-of-function. The authors described two independent pathways for RyR2 gating. The first involves interactions close to the channel pore, which are destabilized upon Ca2+ binding.
The second pathway include a series of conformational transductions from U-motif to S4-S5 linker, which moves out of the way and opens the gate. This is the first paper to solve the fulllength mouse RyR2 structure in both closed and Ca2+ bound open states, both WT and K4593A mutant. This is also the first paper trying to explain the gating of RyR2 in detail using structural approach. The study is important and meaningful; the manuscript is well-written, but I feel that some clarification is needed and some small mistakes need to be corrected. Specifically:

Could the authors explain in more detail about what is the cause of the U-motif compaction?
In  To explain the above motions more precisely, the following sentences are added in the section "U-motif plays a key role in stabilizing the channel in the closed state".
"Upon binding of Ca 2+ to CTD, the upper part of S6cyto rotated together with CTD (Fig. 1g, 4g).
Along with the rotation, S6cyto pushed the N-terminal helix of U-motif, thereby, the parallel shift of the N-terminal helix occurred." (lines 229-231) We also found mistakes in the direction of arrows Fig. 4g, 5h, Supporting Fig. 6f (right). In all cases, the direction of S6cyto should be slightly oriented toward the N-terminal helix of U-motif, therefore, we corrected them.
[Answer to "why in the K4593A mutant there is no rotation of the complex but rather only the

CTD is moved after Ca 2+ binding?"]
CTD is placed between 1-turn b sheet and U-motif ( Supplementary Fig. 9f). Since U-motif, CTD, and S6cyto rotated together upon Ca 2+ binding, it is reasonable that structures of these regions in closed and open states matched well ( Supplementary Fig. 9f left).
The independent rotation of CTD in K4593A indicates that CTD placed between 1-turn b sheet and U-motif has some degree of freedom in the rotation in the K4593A mutant, so that S6cyto, connected just before CTD, the upper part of S6cyto, rotated together with CTD. However, since the compaction of U-motif cannot stably form in K4593A, the rotation cannot proceed further due to collision with the N-terminal helix of U-motif.
To explain more detail about this issue, we added new figures (Supplementary Fig. 9f) and the following sentences at the end of section "Structural basis of the loss-of-function mutation".
"It is interesting that the rotation due to Ca 2+ binding occurred only in CTD and the upper part of D6cyto ( Fig. 5c, g, Supplementary Fig. 9f). Since CTD is placed between 1-turn b sheet connected just before U-motif, (Supplementary Fig. 9f), U-motif, CTD, and S6cyto are supposed to rotate together upon Ca 2+ binding as observed in the open state (Fig. 1f, Fig. 5c, g, Supplementary Fig.   9f). The independent movement of CTD in K4593A indicates that CTD placed between 1-turn b sheet and U-motif have some degree of freedom in the rotation. The upper part of S6cyto rotated together with CTD because of a direct connection to CTD ( Supplementary Fig. 9f). However, the rotation throughout S6 was blocked by the collision with the uncompacted U-motif in K4593A ( Supplementary Fig. 9f)." (lines 290-297) [For your statement "The only interaction between CTD and U-motif seems to be the hydrophobic interaction between F4888 in the CTD and the hydrophobic pocket in the U-motif."] We are afraid that you may misunderstand the interaction between CTD and U-motif. We do not state that the hydrophobic interaction between F4888 in the CTD and the hydrophobic pocket in the U-motif is the only interaction between CTF and U-motif. We showed that F4888A lacking this interaction still had activity, and, moreover, no additive effects were observed with N4177A and F4888A ( Supplementary Fig. 7b). The result from the double mutant indicates that Umotif/S6cyto and U-motif/CTD interactions are involved in the common pathways, and the interaction between CTD and U-motif via F4888 may work as a negative regulator. To avoid further confusion and to clearly show that the roles of the two interactions are different, we modified the number of the line between CTD and U-motif from one to two in Fig. 6b-d.
We also added the following sentences in the Discussion section.
"In addition to direct regulation of the channel gate, S6cyto interacts with U-motif to restrict its movement toward the S2-S3 linker domain (blue T-shaped line, Fig. 6b, left). This negative regulation by S6cyto is supported by hydrophobic interaction between CTD (F4888) and U-motif (dotted line, Fig. 6b, left). Since the interaction is still maintained at the open state, the WT channel is not fully activated (Fig. 6b,  We apologize for this mistake in the legend for Fig. 6a. It is now corrected to purple and green.
We revised Fig. 6b, c, d, and F4888A is now explained as mutation #7 in Fig. 6d. We apologize for this mistake. These are now corrected.
We apologize for this mistake. It is now corrected.
7. Whenever we encounter a LOF mutation, there is always a possibility that the channels are misfolded/did not go to the membrane. Could the authors prove that all the LOF mutants that did not show any signal in the ryanodine binding assays are correctly folded channels? If not, could the authors explain why they think it is not necessary or not possible prove it?
We added a description "10 mM caffeine, a potent RyR activator, released Ca 2+ from ER in cells expressing K4593A, indicating that it forms a functional channel. These findings confirm a lossof-function of the K4593A channel. Similar results were obtained with other substitutions in S4167, E4198, and K4593 (Fig. 2g, Supplementary Table 2)." (lines 145-146)

Could the authors explain why different 3D classes were found during Cryo-EM data
processing if "no major differences were observed among the classes"? (Line91) Different classes during Cryo-EM data processing might correspond to the thermal fluctuations of the molecule. Therefore, it is quite reasonable that there are slight differences among them.
Although the structure in the closed state is classified into 2 classes ( Supplementary Fig. 1f), all the essential interactions discussed in this manuscript are maintained among them. The above situation was essentially the same in the structure in the open state ( Supplementary Fig. 1f). To show that the different 3D reconstructions coming from the same conditions are consistent among them, we now added supplementary figures illustrating reproducibility of their density maps for the most relevant residues ( Supplementary Fig. 3b, 4d, h, 6a).

It would be helpful if the authors could show the distances between atoms when highlighting interactions in atomic models.
In the figures, we added the distances of all highlighting interactions in atomic models.
10. The authors hypothesized that there are two independent pathways for channel opening. If the authors could make a double GOF mutant from each of the pathways (e.g. D4744A and V4879) and use functional experiments to show that there is an additive effect or even spontaneous channel opening, that would be interesting and would make the argument stronger.
Thank you very much for your nice proposal. We are also interested in the two pathways. We are currently testing it by several double mutant channels and planning to present the results with the structures of GOF mutants in the next paper.